Method and composition for chemical synthesis using high boiling point organic solvents to control evaporation

ABSTRACT

A method for reducing evaporation of a liquid reagent solution during solid phase, micro-scale chemical synthesis of a molecule comprising sub-units on an open environment solid support surface. The method includes the steps of providing an open solid support surface including at least one binding site which is functionalized with a reactive chemical moiety; and depositing a substantially controlled and minute volume of liquid reagent solution onto the support surface, and in contact with the binding site. The reagent solution includes reactants contained in at least one relatively high boiling point solvent, in contrast to standard organic solvents for such reagents. Application of a high boiling point solvent substantially reduces evaporation of the reagent solution in the open environment during synthesis on the solid support while enabling the maintenance of a substantially high reaction yield.

TECHNICAL FIELD

This present invention relates, generally, to chemical reactions usinghigh boiling point organic solvents on a support surface, and, moreparticularly, relates to solid phase synthesis at small, open,individual reactive sites spatially separated on the support surface.

BACKGROUND ART

Determination of the sequence of DNA, RNA, and peptide fragmentscontinues to play a significant role in the development of diagnosticmedicine, forensics, molecular biology research, and pharmaceuticalpharmacogenetics. However, more recently attention has turned from thedetermination of sequence itself to identification of the function ofsequences in biochemical pathways and disease states. Because thegenetic influence on most biochemical pathways has been more complexthan originally thought, typically involving multiple genes, multiplemutations in genes, and complex interactions, the need to improve theproductivity to perform simultaneously multiple assays of DNA sequencehas grown.

One way to achieve many parallel simultaneous measurements is to lay outa large number of DNA, RNA, or peptide probe assays onto a microarraywhich can then be probed simultaneously by complex biological samples.Each individual probe in the array, through hybridization (specificbonding), or not, with, for example, DNA or RNA in an unknown sample,provides information about the presence or absence of sequences in thesample.

These types of assays typically test for the presence of a specificnucleic acid sequence, usually a DNA or RNA sequence, although otherspecific binding assays are also possible. As is well known in thefield, this is accomplished by utilizing oligonucleotides synthesizedwith specific, predetermined sequences of interest. Typically suchspecific sequence is based on searches of genome or mutation databasesor, for example, homology to a known or putative gene or amino acidsequence, or catalogued mutations of such sequences. The presence orabsence of many sequences can then be ascertained simultaneously byhybridization under conditions which allow only perfectly or closelymatching sequences to associate.

There are numerous examples of important multiple assays performed bysimultaneous microarray analysis. To improve productivity in diseasediagnosis, an array can be made in which 500 different probes, each onecorresponding to a mutation known to cause, for example, CysticFibrosis, are hybridized with a patient's DNA such that, if any of thecausative mutations are present, that specific feature of the arrayrepresenting that mutation becomes fluorescent. In another usefulexample, DNA sequences corresponding to many genes whose functions areunknown are formed into a microarray. Messenger RNA prepared from bothnormal and diseased tissue samples can then be compared by measuringdifferential intensity of probe hybridization on the many differentsequences corresponding to many different genes simultaneously. Thosegenes hybridized differently in the disease tissue compared to thenormal tissue can then be implicated in the disease pathway, andassigned a function. Additionally there are numerous examples inmolecular biology and pharmaceutical discovery in which the presence orabsence of large number DNA sequences need to be analyzed to determineimportant specifics of a disease state, e.g., resistance to antibiotics,genotype indicative of severity, etc.

Finally, there are many applications in which drug/receptor interactionscan be determined by tethering the candidate drugs (such as smallorganic molecules) or biological receptors to a microarray surface andobserving the degree to which the two associate.

Thus, it is often necessary to create a large number of related, butdistinct chemical features on a microarray. Synthesis of arrays of boundoligonucleotides or peptides is also generally known in the art. In oneapproach to parallel synthesis, known as the T-bag method or diskdesign, an array of individual packets or disks of solid support beadsare physically sorted into four (4) amidite subsets for treatment withthe selected amidite. After each packet of beads has been treated withthe common reagent, the packets must again be manually resorted into thefour subsets for the subsequent synthesis cycle. Such sorting andresorting becomes too burdensome and labor intensive for the preparationof large arrays of oligonucleotides.

Another array approach for the synthesis of support-boundoligonucleotides is that of Southern et. al., U.S. Pat. No. 5,436,327,which performs the synthesis in a very narrow gap between two glassplates. Not only is this technique impractical and cumbersome inpractice since a plurality of different reagents must be appliedaccurately to specific sites on the glass surface, but this approachdoes not allow a continuous synthesis of oligonucleotides. Moreover,since Southern uses standard reagents for phosphoramidite synthesis, thetechnique needs to be performed in a closed environment to prevent rapidevaporation of the highly volatile solvents (E.g., acetonitrile anddichloromethane, as will be described in greater detail below).

One preferred approach for synthesis of arrays of oligonucleotides onopen solid support surfaces is described by Brennan, U.S. Pat. No.5,474,796, which controls delivery of specific reagents throughdrop-on-demand inkjet devices. Brennan provides a general method forconducting a large number of chemical reactions on a support surfacewhere very minute volumes of solutions of chemical reactants are addedto functionalized binding sites on the support surface by means of apiezo electric pump. The functional binding sites are separated fromeach other by the use of a non-functionalized coating material withdifferent surface tension.

While Brennan does not specify the nature of the solvents employed withthis system in order to make this open surface method applicable togeneral chemical synthesis, in practice, most chemical reactions areperformed in highly volatile, low-boiling solvents such as acetonitrileor dichloromethane. One problem associated with using these conventionalsolvents in open synthesis arrangements is that when the delivered dropsare too small, the solvents tend to evaporate too quickly. This isespecially true with the piezo electric delivery pump devices, asapplied in Brennan, since the volume of delivered drops are typicallybetween about 20 picoliters to 2 microliters. In this minute size range,the vapor pressure and surface area to total volume ratio of the dropsare so high that these standard high yield DNA synthesis solventsevaporate before reacting completely.

The rate of evaporation is a function of the surface area of the drop,and is related to 1/R² (where R is the radius of the drop), i.e. thesmaller the drop, the faster the rate of evaporation. At 23° C., a 100micron droplet of acetonitrile (ACN) will evaporate in flight within 1cm of travel. Once the solvent has evaporated, the amidite couplingreaction essentially ceases in the thick, gummy or crystalline residue.

In conventional solid phase oligonucleotide synthesis on controlled poreglass (CPG), for example, the preferred solvent for the tetrazoleactivated, dimethoxytrityl protected nucleotide phosphoramidite couplingstep is acetonitrile. This solvent has been determined to be farsuperior to other common solvent types for phoshoramidite coupling, suchas tetrahydrofuran, dimethoxyethane and nitromethane. Acetonitrilepossesses the ideal combination of acidity, viscosity, dielectricconstant solubility and other properties to promote high (>99%) yieldcoupling. A stepwise coupling yield less than 97% would produce auseless mixture of truncated and/or deleted products.

Acetonitrile (ACN) unfortunately has a rather low boiling point (81°C.), and drops on a support surface evaporate very quickly in an opensystem. The other above-indicated commonly employed solvents for DNAsynthesis (i.e., tetrahydrofuran, dimethoxyethane and nitromethane) alsohave similar boiling points and rates of evaporation as acetonitrile.Accordingly, these solvents tend to evaporate very quickly in opensynthesis systems as well.

One solution would be to deliver a rapid series of droplets of reagentto build up a larger volume for the larger diameter reaction sites.However, this technique becomes ineffective for diameters less thanabout 500 microns, and diameters greater than 500 microns are far toolarge in many array approaches Moreover, the rate of evaporation may beslowed by lowering the temperature of the solvent and reaction surface,but the rate of the coupling reaction may be severely reduced. Finally,the rate of evaporation of a drop can also be slowed by increasing therelative humidity or saturation of the head space vapor by ACN. Inpractice however, stable ACN humidity control is difficult to achieve,and the synthesis device tends to function as a cloud chamber.

DISCLOSURE OF INVENTION

Accordingly, it is an object of the present invention to provide amethod and composition for synthesis of materials on open surfaces whichsubstantially reduces evaporation of the reagent solvent when deliveredin minute volumes thereof.

It is another object of the present invention is to provide a method andcomposition which maintains a substantially high reaction yield.

Another object of the present invention is to provide a method andcomposition of reagent solution which may be delivered throughconventional drop-on-demand delivery assembly.

In accordance with the foregoing objects, the present invention providesa method of reducing evaporation of a liquid reagent solution duringsolid phase, micro-scale chemical synthesis of a molecule comprisingsub-units on an open environment solid support surface. The methodincludes the steps of: (A) providing an open solid support surfaceincluding at least one binding site which is functionalized with areactive chemical moiety; and (B) depositing a substantially controlledand minute volume of liquid reagent solution onto the support surface,and in contact with the binding site. According to the presentinvention, the reagent solution includes reactants contained in at leastone relatively high boiling point solvent, in contrast to standardorganic solvents for such reagents. Such a high boiling point solventsubstantially reduces evaporation of the reagent solution in the openenvironment during synthesis on the solid support while maintaining asubstantially high reaction yield.

In another aspect of the present invention, a method for synthesizing anarray of materials on open surfaces is provided which includes the stepsof: providing a substantially planar, open solid support surfacecontaining an array functionalized binding sites. Each binding site isseparated by surface tension barriers of non-reactive, hydrophobicmaterials. The next step includes depositing a substantially controlledand minute volume liquid reagent solution onto the support surface ateach functionalized binding site for contact with at least one sub-unitof the molecule affixed to the respective binding site. Each reagentsolution includes nucleoside reagents contained in a polar aproticsolvents having a boiling point of at least about 140° C. Due to theelevated boiling point, the evaporation of the minute volumes of reagentsolutions are substantially reduced in an open environment duringmolecule growth. Further, the high coupling yield is substantiallymaintained.

The depositing step may be performed using drop-on-demand delivery ofthe reagent solution. This delivery may be provided through conventionalvalve delivery means or through a piezo-electric pump device, as long asthe delivery volume, about 20 picoliters to about 2 microliters, iscontrolled and accurate. Further, the depositing step is furtherperformed by discreet, stepwise polymer synthesis at each the bindingsite by the phosphoramidite method.

In yet another aspect of the present invention, a nucleoside reagentsolution is provided for oligonucleotide synthesis in relatively minutevolumes. The reagent solution includes a nucleotide reagent, and a polarorganic, aprotic solvent having a boiling point of at least about 140°C. In the small volumes delivered, evaporation of the reagent solutionin an open environment is substantially reduced during oligonucleotidegrowth while maintaining a substantially high coupling yield.

These aprotic solvents are preferably organic in nature and are selectedfrom the group of dinitriles, glymes, diglymes, triglymes,dimethylformamides (DMF), hexamethyphosphorictriamides (HMPA) andtrimethylphosphates. This group preferably include substantially similaracidity properties as that of an acetonitrile solution. Morespecifically, the group of dinitriles are selected from the groupconsisting essentially of malononitrile, succinonitrile, glutaronitrileand adiponitrile. However, the polar organic aprotic solvents may alsobe selected from the group of mononitriles such as valeronitrile andcapronitrile.

BRIEF DESCRIPTION OF THE DRAWING

The assembly of the present invention has other objects and features ofadvantage which will be more readily apparent from the followingdescription of the best mode of carrying out the invention and theappended claims, when taken in conjunction with the accompanyingdrawing, in which:

FIG. 1 is top perspective schematic view of a synthesis apparatusdepositing the reagent solution of the present invention into an arrayof functionalized binding sites of a support surface.

FIGS. 2A and 2B are enlarged side elevation views, in cross-section, ofa support surface and a functionalized binding site illustrating thesurface tension wall effect at the dot-interstices interface. FIG. 2Aillustrates that a micro-droplet reagent solution is deposited on afunctionalized binding site. FIG. 2B illustrates that due to differencesin wetting properties between the reagent solution and the surroundingsurface, the reagent solution beads on the functionalized binding site.

BEST MODE OF CARRYING OUT THE INVENTION

While the present invention will be described with reference to a fewspecific embodiments, the description is illustrative of the inventionand is not to be construed as limiting the invention. Variousmodifications to the present invention can be made to the preferredembodiments by those skilled in the art without departing from the truespirit and scope of the invention as defined by the appended claims. Itwill be noted here that for a better understanding, like components aredesignated by like reference numerals throughout the various figures.

It will further be understood that while the present invention isparticularly suitable for building sequence defined oligonucleotides,the method and composition of the present invention may be employed forsynthesis of any molecule comprising sub-units, and particularlysequential additional synthesis. Hence, the term “sequential unit” or“sub-unit” will be defined as a moiety that is bound to other moietiesof the same or a different kind to form a more complex molecule, such asoligonucleotides and peptide chains. It will further be appreciated thatthe term “open environment” will be defined as any environment which isnot subjected to a closed or vapor saturated system to controlevaporation of volatile solvents.

Attention is now directed to FIGS. 1 and 2 where a solid phase,micro-scale synthesis apparatus, generally designated 10, is shown forbuilding molecules comprising sub-units through the application of themethod and composition of the present invention by sequentially addingsub-units or sequential units to an open, solid support surface 11 in aliquid reagent solution 12. The synthesis apparatus, to be discussed ingreater detail below, is configured to accurately deposit small orminute droplets 13 of reagent solution to functionalized binding sites14 on the solid support surface 11 which are subjected to openenvironmental influences. In accordance with the present invention, thedeposited reagent solution 12 includes chemical reactants contained in ahigh boiling point, polar, aprotic, organic solvents which facilitatesthe control of evaporation thereof during the chemical reaction. Unlikethe prior art standard low boiling point solvents currently employed foropen systems, these high boiling point solvents substantially reduceevaporation of the small reagent solution droplets from the exposedsupport surface 11 so that molecule growth, such as amidite coupling,can be completed. Thus, in an open environment, molecular synthesis canbe more fully prolonged, while maintaining high reaction yields, withoutthe need of either a closed or vapor saturated system to controlevaporation of volatile solvents. As mentioned, once the solvents haveevaporated, coupling reaction essentially ceases in the thick, gummy orcrystalline residue.

In general, solvent systems developed for open environment molecularsynthesis often involve relatively low boiling point solvents.Accordingly, by substituting these low boiling point solvents withrelatively high boiling point solvents which exhibit similar solventproperties to the replaced solvents, to be discussed, the openenvironment reaction may be completed without the necessity ofcontrolling evaporation of these solvents. While the present inventionmay be generally applied to any molecular synthesis where low boilingpoint solvents may be problematic in an open environment, the relativelyhigh boiling point solvent substitution has been found particularlyuseful and beneficial for organic molecular synthesis and forbiopolymers, such as oligonucleotides, peptides and peptide nucleicacids.

In the preferred form, the boiling point of the polar organic solventsis at least about 140° C. at one (1) atmosphere (atm), as opposed to alower boiling point of about 81° C. for the standard DNA solvent,acetonitrile. Thus, it will be appreciated that a relatively highboiling point will be defined as a boiling point of the solvent of atleast about 140° C. at one (1) atm, while a relatively low boiling pointwill be defined as a boiling point of not more than about 100° C. at one(1) atm for the standard DNA solvents of acetonitrile, tetrahydrofuran,dimethoxyethane and nitromethane, as well as other low boiling pointsolvents such as ethanol, acetone and pyridine.

This higher boiling point assures that when minute or small volumedroplets are applied in an open environment, typically in the size rangeof between about twenty (20) picoliters to about two (2) microliters,the droplets will not evaporate too quickly at normal ambient laboratorysynthesis conditions in an open environment so that the chemicalreaction can be prolonged or completed. Importantly, the lower vaporpressure of the high boiling point solvent when added in a mixture to ahigh vapor pressure solvent, tends to substantially lower the vaporpressure relative that of the prior art standard DNA solvents. Thus,high yield DNA synthesis can be completed before significant evaporativefactors impede the chemical reaction.

Moreover, to promote a stepwise high coupling yield of at least about97% (the conventional DNA standard), these high boiling point solventsystems preferably possess similar acidity, dielectric constant andsolubility properties as that of the standard low boiling point solventsystems in current use. In accordance with the present invention, thesehigher boiling point solvents are not only less volatile than thestandard solvents, but they include similar solvent properties whichmimic the standard solvents so that substantially high reaction yieldscan be maintained. It has been found, for instance, that there are somehigh boiling solvent/activator combinations which provide high-yieldamidite coupling similar to the acetonitrile/tetrazole pair, while alsooffering the reduced rate of evaporation necessary for surface arraysynthesis in small drops. The relative evaporation times of differentcandidate solvents compared to one standard DNA solvent, acetonitrile,have been significantly increased by a factor of at least about fifty(50).

The most preferred high boiling point, polar, aprotic, organic solventshave been determined to be from the group of dinitriles, specificallymalononitrile, succinonitrile, glutaronitrile, adiponitrile andpimelonitrile. Compared to the low boiling point of acetonitrile (81°C.), the boiling point of these solvents is substantially higher at 218°C. for malononitrile, 265° C. for succinonitrile, 286° C. forglutaronitrile, 298° C. for adiponitrile and 310° C. for pimelonitrile.Hence, more preferably, the relative high boiling point will be at leastabout 200° C. at one (1) atm.

Moreover, the vapor pressures of these solvent systems are substantiallylower than that of the more volatile standard DNA solvents (e.g., 70 mmof acetonitrile at room temperature). The vapor pressure and surfacearea to total volume ratio for a fifty (50) picoliter reagent solutiondroplet containing a solvent from the dinitrile group, hence, issignificantly lower than that for a similarly sized droplet containingan acetonitrile solvent.

Accordingly, these solvents evaporate at a much slower rate at roomtemperature or standard synthesis environment. For instance, theevaporation half-life time of a 0.1 microliter drop of a dinitrilesolvent is greater than about one (1) hour, as compared to about ten(10) seconds for acetonitrile. Hence, under a similar reactionenvironment, detrimental evaporative influences during polymer synthesisare less likely in the present invention.

Importantly and as set forth above, these solvent systems possesssimilar solvent properties as that of acetonitrile for thephosphoramidites such as acidity, viscosity, dielectric constant andsolubility properties. For example, the group of dinitrile/acetonitrileanalogues possesses a similar acidity to acetonitrile, and providesexceptionally high coupling yields (>99%) with the acid catalysts, suchas tetrazole and S-ethyl tetrazole, and standard phosphoramidites.Adiponitrile or glutaronitrile with S-Ethyl tetrazole, as an acidcatalyst, have been found to be the most preferred solvent/activatorpairs.

The mononitrile group of the aprotic organic solvents have also beendetermined to substantially benefit small droplet polymer synthesis inopen environments. These mononitriles solvents include: valeronitrile,having a high boiling point of 141° C.; capronitrile, having a highboiling point 163° C.; and benzonitrile, having a high boiling point190° C. The vapor pressures of these mononitriles at room temperatureare 5.0 mm, 1.0 mm and 0.5 mm, respectively, which is substantiallylower than that of the standard DNA solvents. These mononitriles, hence,evaporate at a much slower rate at room temperature than acetonitrile,having an evaporation half-life time of a 0.1 microliter drop of greaterthan about one (1) hour.

While these mononitriles have been determined to be an inferior solventfor the phosphoramidites as compared to the dinitriles, probably due totheir dominating alkyl residues, the mononitriles still possess similarsolvent properties to acetonitrile for the phosphoramidites (e.g.,acidity).

The polar, aprotic group of oxygenated solvents, such as diethlyeneglycol dimethyl ether (diglyme), and triethylene glycol dimethyl ether(triglyme), have also been determined to be suitable high boiling pointsolvents for polymer synthesis. These oxygenated solvents provide goodsolubility for the phosphoramidites, as well, although are slightly morebasic than the dinitriles. Accordingly, a more acidic catalyst isrequired to obtain higher coupling yields. One such acidic catalyst ispyridine hydrochloride (Py.HCl).

The boiling point of diglyme is 162° C., while that of triglyme is 216°C. Further, the vapor pressures of these oxygenated solvents at roomtemperature are 1.5 mm and 0.5 mm, respectively. Again, since the vaporpressure is lower than that of the standard DNA solvents, theevaporation half-life time of a 0.1 microliter drop is also longer thanthat of the standard DNA solvent. Coupling yields for these solventshave been greater than 98%.

Other high boiling solvents which yield good solubility for thephosphoramidites and more modest coupling yields with Py.HCl than thosesolvents described above are hexamethyl phosphoric triamide (HMPA),N-methyl pyrolidinone (NMP) and dimethylformamide (DMF). These solvents,however, may be better suited in combination with pyridine for the usewith H-phosphonate coupling chemistry.

The method and solvent system of the present invention may also beemployed with other organic synthesis in addition to DNA synthesis. Asset forth in Example 2 below, high boiling point solvents can beutilized in combinatorial synthesis of amino acid hydroxamatederivatives as well which yielded a library of 500 different amino acidhydroxamate sulfonamide derivatives.

The present invention is particularly suitable for application with thedelivery apparatus of Brennan, U.S. Pat. No. 5,474,796 (hereinafter, the'796 Patent) which is incorporated herein by reference in its entirety.Briefly, as shown in FIGS. 1 and 2, open, functionalized reactive sites14 are defined or separated by non-reactive hydrophobic surface tensionbarriers, and specific reagents are delivered to the individual sitesusing drop-on-demand inkjet devices 15. Preferably, the size of eachreactive or functionalized binding site 14 is typically about twenty(20) to about 2000 microns. Solutions of chemical reactants aredelivered to the functionalized binding sites 14 on the support surface11 through a piezoelectric or solenoid nozzle, or any other nozzlessystem capable of controllably dispensing small droplets accurately. Theinitial size of an ejected droplet is determined principally by thenozzle orifice diameter and the viscosity and surface tension of theliquid medium. The typical droplets 13 range in size from abouttwenty-five (25) to about 250 microns.

The reagent solution containing the chemical reactant and the highboiling point, polar, aprotic, organic solvent can be delivered to thefunctionalized binding site 14 through a piezo-electric pump (not shown)in an amount where the solution of chemical reactant at each bindingsite is separate from a reagent solution at other adjacent binding sitesby surface tension. Briefly, in the piezo-electric pump, reagentsolution is inserted through an inlet into a chamber formed between anupper plate and an opposed lower plate of the piezo-electric pump.Application of a voltage difference across the upper and lower platescauses compression of the piezo, forcing a micro-droplet of reagentsolution out through the nozzle.

As best viewed in FIG. 2A, micro-droplet 13 of reagent solution isdeposited on the functionalized binding site 14. Due to the differencesin wetting properties of the reagent solution 12 on the functionalizedbinding site 14 and the surrounding surface 16 (FIG. 2B), themicro-droplet 13 of the reagent solution beads on the functionalizedbinding site 14 and the reactants in solution react with the solidsupport surface.

The piezoelectric pump that may be utilized in the invention deliversminute droplets 13 of liquid to the support surface 11 in a very precisemanner. The picopump design is similar to the pumps used in ink jetprinting, and is capable of producing fifty (50) micron or sixty-five(65) picoliter droplets at up to 3000 Hz.

From the above description of the present apparatus, it will beunderstood that the method of the present invention is provided forreducing evaporation of a liquid reagent solution during solid phase,micro-scale chemical synthesis of a molecule comprising sub-units on anopen environment solid support surface 11. The method includes the stepsof: (A) providing an open solid support surface 11 including at leastone binding site 14 which is functionalized with a reactive chemicalmoiety. The next step includes: (B) depositing a substantiallycontrolled and minute volume of liquid reagent solution 12 onto thesupport surface 11, and in contact with the binding site 14. Inaccordance with the present invention, the reagent solution includesreactants contained in at least one relatively high boiling pointsolvent, in contrast to standard organic solvents for such reagents.Such a high boiling point solvent substantially reduces evaporation ofthe reagent solution in the open environment during synthesis on thesolid support 11 while maintaining a substantially high reaction yield.

The depositing step may be performed using drop-on-demand delivery ofthe reagent solution 12. This delivery may be provided throughconventional delivery means or through a piezo-electric pump device 15,as long as the delivery volume, about twenty (20) picoliters to abouttwo (2) microliters, is controlled and accurate. Further, the depositingstep is further performed by discreet, stepwise synthesis at each thebinding site by the phosphoramidite method or by the H-phosphomatemethod.

In another aspect of the present invention, and more particularly, amethod of solid phase, micro-scale chemical synthesis of anoligonucleotide chain is provided on an open environment solid supportsurface. The method includes the steps of (A) providing an open solidsupport surface 11 including at least one binding site 14 which isfunctionalized with a reactive chemical moiety; and (B) depositing asubstantially controlled and minute volume of liquid reagent solution 12onto the support surface 11, and in contact with the binding site 14. Inthis embodiment, the reagent solution 12 includes reactants contained ina high boiling point, polar, aprotic organic solvent having a boilingpoint of at least about 140° C. This technique substantially reducesevaporation of the reagent solution in the open environment duringoligonucleotide synthesis on the solid support while maintaining asubstantially high coupling yield thereof.

Steps A and B may be repeated using the same or different chemicalreactants to form at least one continuous oligonucleotide chain. Whenthe open support surface 11 includes an array of functionalized reactionsites 14 (FIG. 1), the depositing step may be accomplished byindividually depositing reagent solutions to selected reaction sites 14of the array. Further, the depositing step may be accomplished bydepositing the reagent solution to each selected reaction site 14 in anamount where the reagent solution at each reaction site 14 is separatedfrom the reagent solution at other binding sites by surface tension.This may be performed by providing a support surface of eachfunctionalized reaction site 14 which has a higher surface tensionrelative to the surrounding support surface 16 surrounding eachfunctionalized binding site 14, which are preferably composed ofnon-reactive, hydrophobic materials.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modecontemplated for carrying out various aspects of the invention. It is tobe understood that this example in no way serves to limit the true scopeof the invention, but rather are presented for illustrative purposes.

EXAMPLE 1 Oligonucleotide Synthesis on an Array Plate

Oligonucleotide synthesis was performed on a patterned glass array platewhich provided functionalized aminoalkylsilane spots for synthesisseparated by lipophilic fluoroalkylxysilanes. The array contained 500spots with diameters of 0.5 mm. The array was synthesized, as describedin the '796 Patent, using aminopropyl- andtetradecafluoro-1,1,2,2-tetrahydrooctyl siloxane for primary patterning.

Conversion of the short aminopropyl linker into the long-chainhydroxylalkyl linker was accomplished by treating the array withp-Nitrophenylchloroformate in dioxane:DCM (1:1) for 2 hours. Unreactedaminopropyl groups were capped using a 1:1 mixture of acetic anhydrideand pyridine. The resulting carbamate intermediate was than convertedinto a hydroxyl bearing urea by reaction with 6-aminohexanol inacetonitrile overnight. A cleavable linker was synthesized by treatingthe patterned surface with 5′-DMT-nucleoside-3′ succinate inacetonitrile utilizing TOTU as an activator for 2 hours.

Assembly of oligonucleotides on the so prepared spots was carried outaccording to the standard phosphoramidite procedure. Standardphosphoramidites and S-Ethyl tetrazole were dissolved in a mixture ofadiponitrile (ADN) or glutaronitrile (GLN) and acetonitrile (ACN). Theaddition of a defined amount of the lower-boiling acetonitrile allowedthe control of solvent viscosity and drop formation from the jet. Amixture of 90% ADN and 10% ACN proved optimal for drop formation andviscosity of the reagents.

Delivery of the appropriate protected nucleotides and activatingreagents was directed to individual spots using a micropump apparatus asdescribed in the '796 Patent. All other steps (e.g. DMT deblocking,washing) were performed on the array in a batch process by flooding thesurface with the appropriate reagents. Reagents were then removed fromthe surface by spinning the array at high velocity.

After synthesis, the oligonucleotide was cleaved from the surface anddeprotected by aqueous ammonia. The product (e.g. T10) was analyzed byHPLC and HPCE and showed a quality comparable or better than standardoligonucleotide synthesis on CPG. This leads to the conclusion that thestepwise synthesis yield was greater than about 98%.

EXAMPLE 2 Combinatorial Synthesis of Amino Acid Hydroxamate Derivatives

A patterned array was utilized as described in Example 1. Amino acidswere first coupled to the array and then derivatized with differentsulfonylchlorides to achieve diversity. Fmoc protected amino acids andactivator (HATU) were dissolved in DMF:CH2CL2 (9:1). Both reagents weredirected to single spots as described in the '796 Patent. After 15minutes, the reagents were removed by spinning the array. Aftercoupling, the array was washed with dichloroethane. Next the Fmocprotecting groups of the amino acids were removed by flooding the arraysurface with a 10% solution of piperidine in DMF for 10 minutes. Afterwashing with DMF and THF, different sulfonyl chlorides dissolved inpyridine were directed to individual spots for derivatization. After areaction time of 10 minutes, the reagents were removed by spinning andthe array was washed with pyridine. After a final wash with pyridine,DMF, DMSO and DCE, the synthesized compounds were cleaved with 2Mhydroxylamine in water/dioxane (1:7.5) for 48 hrs in a sealed chamber toyield a library of 500 different amino acid hydroxamate sulfonamidederivatives.

What is claimed is:
 1. A method of reducing the evaporation of a liquidreagent solution during solid phase chemical synthesis of moleculesselected from the group consisting of nucleic acids, peptides andpeptide nucleic acids on an open environment solid support surface, saidmethod comprising the steps of: (a) providing an open solid supportsurface including at least one binding site which is functionalized witha chemical moiety; and (b) depositing a minute volume of liquid reagentsolution onto said support surface, and in contact with said bindingsite, said reagent solution including reactants contained in at leastone polar, aprotic solvent having a boiling point of at least about 140°C. and selected from the group consisting of dinitriles, mononitriles,glymes, diglymes, triglymes, and trimethylphosphates, to reduceevaporation of the reagent solution in the open environment duringsynthesis on said solid support.
 2. The method according to claim 1wherein, the boiling point of said solvent is at least about 200° C. 3.The method according to claim 1 wherein, said solvent is a dinitrilewith an acidity substantially similar to that of an acetonitrilesolution.
 4. The method according to claim 1 wherein, said solvent is adinitrile and is selected from the group consisting of succinonitrile,glutaronitrile, adiponitrile and pimelonitrile.
 5. The method accordingto claim 1 wherein said depositing step is performed by usingdrop-on-demand delivery of said reagent solution.
 6. The methodaccording to claim 5 wherein, said drop-on-demand delivery of saidreagent solution is performed through an ink jet printing device.
 7. Themethod according to claim 5 wherein said drop-on-demand delivery of saidreagent solution is performed through a piezo-electric pump.
 8. Themethod according to claim 1 wherein said solvent is a mononitrile and isselected from the group consisting of valeronitrile, benzonitrile andcapronitrile.
 9. The method according to claim 1 wherein, said moleculesare synthesized by sequentially adding sub-units thereto.
 10. The methodaccording to claim 1 wherein said open support surface is a glasssurface.
 11. The method according to claim 1 wherein said depositingstep is accomplished by individually depositing reagent solutions tosaid binding sites of said array.
 12. The method according to claim 1wherein said solid support surface is substantially planar.
 13. Themethod according to claim 1 wherein the reagent solution at said bindingsite is separated from the reagent solution at other binding sites bysurface tension.
 14. The method according to claim 1 wherein saidfunctionalized binding site has a higher surface tension relative to theareas surrounding said functionalized binding site.
 15. The methodaccording to claim 14 wherein said areas surrounding said functionalizedbinding site are comprised of non-reactive materials.
 16. The methodaccording to claim 1 wherein the volume of the deposited reagentsolution is about 20 picoliters to about 2 microliters.
 17. A method ofsolid phase chemical synthesis of an oligonucleotide chain on an openenvironment solid support surface, said method comprising the steps of:(A) providing an open solid support surface including at least onebinding site which is functionalized with a chemistry moiety; and (B)depositing a minute volume of liquid reagent solution onto said supportsurface, and in contact with said binding site, said reagent solutionincluding reactants contained in a polar, aprotic solvent of at leastabout 140° C. boiling point and selected from the group consisting ofdinitriles, mononitriles, glymes, diglymes, triglymes, andtrimethylphosphates, to reduce evaporation of the reagent solution inthe open environment during oligonucleotide synthesis on said solidsupport.
 18. The method of oligonucleotide synthesis according to claim17 wherein, the boiling point of said solvent is at least about 200° C.19. The method of oligonucleotide synthesis according to claim 17wherein, said solvent is a dinitrile with an acidity substantiallysimilar to that of an acetonitrile solution.
 20. The method ofoligonucleotide synthesis according to claim 17 wherein, said solvent isa dinitrile and is selected from the group consisting of succinonitrile,glutaronitrile, adiponitrile and pimelonitrile.
 21. The method ofoligonucleotide synthesis according to claim 17 wherein, said synthesisis performed by the H-phosphonate method.
 22. The method ofoligonucleotide synthesis according to claim 17 wherein, said solvent isa mononitrile and is selected from the group consisting ofvaleronitrile, benzonitrile and capronitrile.
 23. The method ofoligonucleotide synthesis according to claim 17 wherein, said depositingstep is performed by using drop-on-demand delivery of said reagentsolution.
 24. The method of oligonucleotide synthesis according to claim23 wherein, said drop-on-demand delivery of said reagent solution isperformed through a piezo-electric pump device.
 25. The method ofoligonucleotide synthesis according to claim 17 wherein, said synthesisis performed by the phosphoramidite method.
 26. A nucleoside reagentsolution for oligonucleotide synthesis in minute volumes on an openenvironment solid support surface comprising: a nucleotide reagent; anda polar, aprotic solvent having a boiling point of at least about 140°C. and selected from the group consisting of dinitriles, mononitriles,glymes, diglymes, triglymes, and trimethylphosphates, to reduceevaporation of the reagent solution in an open environment duringoligonucleotide synthesis.
 27. The nucleoside reagent solution accordingto claim 26 wherein, the volume of the reagent solution is about 20picoliters to about 2 microliters.
 28. The nucleoside reagent solutionaccording to claim 27 wherein, the boiling point of said solvent is atleast about 200° C.
 29. The nucleoside reagent solution according toclaim 26 wherein, said solvent is a dinitrile with an aciditysubstantially similar to that of an acetonitrile solution.
 30. Thenucleoside reagent solution according to claim 26 wherein, said solventis a dinitrile and is selected from the group consisting ofmalononitrile, succinonitrile, glutaronitrile, adiponitrile andpimelonitrile.
 31. The nucleoside reagent solution according to claim 26wherein, said solvent is a mononitrile and is selected from the groupconsisting of valeronitrile, benzonitrile and capronitrile.